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Reportedly, CD300f negatively regulates interactions between dendritic and T cells, and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines; an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, co-sedimentation and surface plasmon resonance using phospholipid containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.
The human CD300 family of receptors are Type I transmembrane proteins that contain a single IgV-like extracellular domain. Four members of the family contain a charged residue in their transmembrane domain and associate with ITAM-containing adaptor molecules like DAP12 and FcεRIγ; two other members, CD300a and -f, contain extended cytoplasmic tails with tyrosine based signaling motifs, including ITIMs. Mouse CLM-1 appears to be the functional orthologue of CD300f, as they have in common consensus ITIM motifs and the ability to bind phosphatases (1, 2).
CD300 molecules are expressed on leukocytes and they can positively or negatively augment cellular responses, depending on the character of their signaling motifs (2). Most of this information has been obtained by Ab cross-linking, as essentially nothing is known about the ligands. Mouse CD300f was first described as an in vitro negative regulator of osteoclastogenesis (1). Its expression is largely confined to myeloid cells (1, 3, 4), where in vitro cross-linking studies indicate it can serve as an inhibitory or activating receptor (1, 5, 6) and can mediate caspase-independent cell death (7). Using CD300f deficient mice in a multiple sclerosis mouse model, it was shown that CD300f acts as a negative regulator of myeloid cell activity by suppressing demyelination, and the release of inflammatory cytokines (4). Blockade of CD300f recognition enhanced DC-initiated T-cell proliferation and antigen-specific T-cell responses both in vitro and in vivo (3) and indicated that T cells expressed a CD300f- specific ligand.
We show that a CD300f/Fc chimeric protein binds activated T lymphocytes, as well as apoptotic lymphocytes and insect cells, indicating evolutionary conservation of the ligand, and that the binding can be inhibited by Annexin V. Using a variety of approaches with phospholipid containing liposomes, we show that CD300f preferentially binds phosphatidylserine and that its expression can enhance phagocytosis.
We created a chimeric protein with the extracellular part of mouse CD300f fused to human Fc (CD300f/Fc) (Supplemental Table I); hLAIR-1R65K/Fc (LAIR-1/Fc) was used as a negative control (8). They were purified from culture supernatants by protein A-Sepharose columns.
T cells (>98% pure) were isolated from spleens of C57BL/6 mice by negative selection (MACS MicroBeads, Miltenyi Biotec). After blocking Fc receptors, cells were stained with AlexaFluor488-conjugated chimeric proteins for 1 h at RT and analyzed by flow cytometry. For binding inhibition, the cells were incubated with 10 μg of Annexin V (BD) for 30 min before staining.
Abs used: CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7) and CD69 (H1.2F3), conjugated to FITC, PE, PE-Cy5.5, PE-Cy7 or APC. 7-AAD was used to detect dead cell populations. Data were collected on BD FACSort flow cytometer using Cell Quest and analyzed with FlowJo.
Synthetic 1-palmitoyl-2-oleoyl (PO) phosphatidylserine (PS), (PO)phosphatidylglycerol (PG), 1, 2-dioleyol phosphatidylcholine (DOPC), and bovine liver L-α-phosphatidylinositol (PI) were from Avanti Polar Lipids. Liposomes were prepared by evaporating the chloroform from the desired phospholipid mixture with N2 gas. Large multi-lamellar vesicles were formed by swirling in 10 mM HEPES pH 7.4 with 140 mM NaCl. Small unilamellar vesicles were prepared by sonication for 10 min on ice with minute gap intervals. Large vesicles were removed by 45μ filters.
1 mM liposomes were immobilized on L1 a sensor chip and blocked with BSA. CD300f/Fc was injected and its binding recorded. After dissociation of mCD300f with 2.5M NaCl + 5 mM EDTA, AnnexinV was passed through to block the binding; the unbound AnnexinV was removed with running buffer (10mM HEPES pH 7.4, 140mM NaCl and 2.5 mM CaCl2). CD300f/Fc protein was injected again and binding to the L1-PS sensor that was blocked by AnnexinV was recorded. Data were analyzed by BIACORE 3000 and BIAevaluation Software.
The complex formed by liposomes and binding proteins localizes in the pellet fraction, whereas proteins not binding the liposomes remain in the supernatant fraction. The PS:PC (4:1), and PC:PS (4:1) liposomes (0.5 mM) were incubated with 25 μg/ml of CD300f/Fc protein for 45 min at RT and centrifuged at 100,000 × g for 1h. The pellet fractions and supernatant fractions (precipitated with 10% TCA) were re-suspended in loading buffer, resolved by SDS-PAGE and stained with Coomassie Blue.
1 mM liposomes in HBS-buffered saline were incubated in 96-well maxisorp plates at 4°C overnight. The wells were blocked with 2% BSA for 2h at RT and incubated with CD300f/Fc (15 μg/ml) for 2h at RT. The plate was washed and incubated with anti-human Ig Ab coupled to HRP (Jackson ImmunoResearch) for 1h at RT before measuring absorbance at 450 nm.
Mouse fibroblast L929 cells were used for phagocytic assays (9). Cells were transfected with an empty vector (pcDNA3.1+) or vector encoding mouse CD300f (Supplemental Table I). Thymocytes isolated from C57BL/6 mice were incubated for 6 h at 37°C with 10μM dexamethasone (Sigma), labeled with TFL-4 (Oncoimmunin), washed and added to L929 cells cultured on glass dishes at a ratio of 5:1. After incubation, L929 cells were washed, treated with trypsin-EDTA, fixed and analyzed by flow cytometry. For microscopy, L929 cells were washed, fixed and visualized by Zeiss LSM710 laser-scanning confocal microscope. The images were obtained using 63X Plan-Apochromat objective and Zeiss ZEN software. To verify the engulfment of apoptotic cells by L929 cells images were obtained along the z-axis. A representative single optical section is shown in the figure. Animal study protocols were reviewed and approved by the NIAID Animal Care and Use Committee.
We found that CD300f/Fc, but not LAIR-1/Fc, preferentially bound to splenocytes with low FSC and SSC (R1) (Fig. 1A), indicating that CD300f recognizes cells that are apoptotic or dead. Induction of cell death by starvation or gamma irradiation resulted in increased specific binding of CD300f/Fc to 7-AAD-positive R1.1 cells (Fig. 1B), as well as T and B cell lines (Supplemental Fig. 1A). The CD300f/Fc fusion protein also bound effectively to apoptotic insect SC2 cells (Supplemental Fig. 1B), demonstrating that the ligand for CD300f is conserved among evolutionary distant species.
Although CD300f/Fc protein bound strongly to apoptotic/dying splenocytes, some binding of the chimeric protein to 7-AAD-negative cells was also observed (Fig. 1A), indicating that the ligand for CD300f can be expressed on the surface of live cells. CD300f/Fc bound specifically to OVA-activated (CD69+) CD4 and CD8 splenocytes from OVA-immunized mice and that there was virtually no interaction with the non-activated (CD69−) population of T cells (Fig. 2A). A hallmark of cell death is the transfer of phospholipids, such as phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane (10); however, the appearance of PS on the cell surface is not restricted to apoptosis and some cells (e.g. T and B cells) display PS on the cell surface in response to activation (11, 12). Since CD300f/Fc bound to apoptotic and activated T cells, as well as dead cells, we hypothesized that the phospholipid PS could be the ligand for CD300f. Therefore, we isolated splenic CD4 T cells, stimulated them with PMA/ionomycin and investigated the binding of CD300f/Fc to the cells treated or not with Annexin V, a protein known to specifically bind PS (13). Following PMA/ionomycin treatment, we analyzed both apoptotic (7-AAD+) and non-apoptotic (7-AAD−) CD4 T cell populations (Fig. 2B). In agreement with previous results (Fig. 1), CD300f/Fc bound to a pool of apoptotic T cells. Importantly, the pretreatment of cells with Annexin V lead to a 2-fold decrease of CD300f binding (Fig. 2B). Stimulation with PMA/ionomycin resulted in activation of a portion of live T cells (7-AAD−CD69+) and subsequent binding of CD300f/Fc. Similarly to apoptotic cells, pretreatment of activated cells with Annexin V resulted in 50% inhibition of CD300f binding. The marginal binding of the CD300f/Fc to the 7-AAD− CD69− CD4 T cell population was also blocked by Annexin V.
The ability of Annexin V to block binding of CD300f/Fc to apoptotic or activated T cells (Fig. 2) suggested that PS is the ligand for CD300f. Since binding of Annexin V and other proteins to PS is dependent on Ca2+, we investigated whether CD300f binding to dead cells also required a metal ion. The presence of EDTA or EGTA chelating agents abolished the binding of CD300f/Fc to 7-AAD+ cells, indicating the requirement for metal ions, likely Ca2+ (Fig. 3A). We directly assessed the binding of CD300f to PS with liposomes containing different phospholipids by ELISA and surface plasmon resonance (SPR). While the LAIR-1/Fc did not bind any liposomes, CD300f fusion protein bound predominantly to liposomes containing PS and binding correlated with the level of PS present in the assay (Fig. 3B). Additionally, CD300f, but not LAIR-1/Fc, was found to associate with liposomes containing PS in a lipid sedimentation assay. Increasing the concentration of PS in the liposomes resulted in a larger amount of CD300f/Fc co-sedimenting with the liposomes (Fig. 3C). Finally, using SPR, we demonstrated that CD300f/Fc bound very efficiently to PS-containing liposomes. Importantly, after removal of CD300f/Fc bound to the PS and subsequent blocking of PS by Annexin V, the binding of CD300f/Fc was substantially reduced (Fig. 3D), further demonstrating that PS is a specific ligand of CD300f. We also found that Annexin V inhibited binding of CD300f/Fc to pure PS or dead cells in a dose-dependent manner (Supplemental Fig. 1C, D).
Since PS exposure on the cell surface can act as an “eat me” signal (14) and the clearance of dead cells is important for immune homeostasis (15), we investigated whether the presence of CD300f affects phagocytosis of apoptotic cells. We transfected mouse L929 cells with CD300f and analyzed the ability of the transfected cells to phagocytose apoptotic thymocytes. The cells expressing CD300f phagocytosed between 30–50% more apoptotic cells compared to control-transfected L929 cells (Fig. 4A, B). The presence of soluble CD300f/Fc, but not LAIR-1/Fc, interfered with uptake of the apoptotic cells by CD300f transfected cells (data not shown). Examination by confocal microscopy verified that phagocytosis and not adherence was occurring; moreover, contrary to the mock-transfected cells, CD300f transfected cells often contained several apoptotic cells (Fig. 4C). We obtained similar results with NIH3T3 cells expressing CD300f (not shown). These results show that recognition of apoptotic cells by CD300f can have a stimulatory role in aiding the clearance of apoptotic cells, in agreement with studies demonstrating that CD300f can provide activation signals (6, 7). In addition to its ITIM motifs, the cytoplasmic tail of mCD300f has an ITSM switch motif and two other tyrosines, one of which may be involved in PI3K binding (7). Accordingly, future studies will be directed toward elucidating the molecular basis for the observed enhanced phagocytosis.
The online version of this article contains supplemental material.